Chlorella sorokiniana rbcL基因的克隆与序列分析及其与18S rRNA基因在分类学上的比较

Cloning and Sequence Analysis of rbcL Gene from Chlorella Sorokiniana and Comparison with 18S rDNA Gene in Taxonomy

Rubisco大亚基基因(rbcL)广泛用于系统学分析中,在本文中以Chlorella sorokiniana CS-01叶绿体基因组DNA为模板,PCR扩增rbcL全长编码区序列,序列分析表明:该片段全长1428 bp,其中包括1425 bp的编码区序列,编码475个氨基酸,经BLAST比对发现同源性最高的为Chlorella sp.IFRPD 1018,同源性达到99.2%。同时构建系统发育树,结果显示Chlorellasorokiniana CS-01与Chlorella sp.IFRPD 1018在同一分支中。18S rDNA序列分析表明:该片段全长1740 bp,经BLAST比对发现同源性最高的为Chlorella sorokiniana,同源性达到99.7%,构建系统发育树显示Chlorella sorokiniana CS-01与两株Chlorellasorokiniana在同一分支中,支持率达到100%。但是18S rDNA序列构建的系统发育树鉴定的主要分支很少,即使鉴定出分支,该分支的支持率也比较弱,而rbcL基因序列构建的系统发育树则分支清晰且支持率较高。可见18S rDNA序列比rbcL基因序列保守性更强,进化速度更慢,因此rbcL基因序列适合亲缘关系更近的微藻的系统研究。

In this paper,the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene rbcL was cloned and analyzed from Chlorella sorokiniana CS-01 chloroplast genome.Sequencing analysis indicated that this fragment was 1428 bp in size.The com-plete open reading frame of Chlorella sorokiniana CS-01 rbcL gene is 1425bp encoding a polypeptide of 475 amino acids.The BLAST result shows that Chlorella sorokiniana CS-01 has the highest homologies with Chlorella sp.IFRPD 1018 with 99.2% sequence similarity.The BLAST result of 18S rDNA shows that Chlorella sorokiniana CS-01 has the highest homologies with Chlorella sorokiniana with 99.7% sequence similarity.Two phylogenetic trees of rbcL and 18S rDNA were constructed with the published rbcL and 18S rDNA se-quences of the relative microalga species.However,in the phylogenetic tree of 18S rDNA,the main branches are small,even if the branches were identified,the bootstrap values of branches are relatively weak,while in the phylogenetic tree of rbcL gene,the main branches are clear with high bootstrap values.This suggests that 18S rDNA gene is more conservative and evolves much slower than rbcL genes.Thus,using rbcL sequences in molecular systematics seems to be more appropriate for the phylogenetic analysis of closely related microalga than 18S rDNA.